S1

1 Supplementary Information

2

3 Interaction of CuO Nanoparticles with Plant Cells: Internalization, Oxidative

4 stress, Electron Transport Chain Disruption, and Toxicogenomic Responses

5 Yanhui Dai,1 Zhenyu Wang,2,3 Jian Zhao,1 Lili Xu,1 Lina Xu,1 Xiaoyu Yu,1 Yongpeng Wei,1

6 and Baoshan Xing*,4

7

8 1 Institute of Costal Environmental Pollution Control, and Ministry of Education Key Laboratory

9 of Marine Environment and Ecology, Ocean University of China, Qingdao 266100, China

10 2 Institute of Environmental Processes and Pollution Control, and School of Environmental and

11 Civil Engineering, Jiangnan University, Wuxi 214122, China

12 3 Laboratory for Marine Ecology and Environmental Science, Qingdao National Laboratory for

13 Marine Science and Technology, Qingdao 266071, China

14 4 Stockbridge School of Agriculture, University of Massachusetts, Amherst, Massachusetts 01003,

15 United States

16

17 *Corresponding author

18 Tel.: +1 413 545 5212; fax: +1 413 577 0242

19 E-mail address: bx@umass.edu (Dr. Baoshan Xing)

20

21

22

23

24

25

Electronic Supplementary Material (ESI) for Environmental Science: Nano.This journal is © The Royal Society of Chemistry 2018

S2

26 Materials and methods27 Reactive oxygen species determination

28 The hydrogen peroxide (H2O2) was detected based on reacting with ammonium

29 molybdate to produce a peroxomolybdic acid complex. H2O2 content was performed

30 using a hydrogen peroxide assay kit (A064, Jiancheng, Nanjing, China). The coefficient

31 of variation (CV) was 1.7%. Absorbance was determined at 405 nm by a microplate

32 reader (Thermo, USA).

33 Hydroxyl radical (OH·) was determined using a hydroxyl free radial assay kit (A018,

34 CV=1.4%, Jiancheng, Nanjing, China) based on the reaction of H2O2 with Fenton

35 reaction. A colouration reaction was generated following the addition of the electron

36 acceptor and Griess reagent. The absorbance was determined using a microplate reader

37 (Thermo, USA) at 550 nm.

38 Superoxide anion (O2·-) was determined by inhibition and produce4 produce

39 superoxide anion assay kit (A052, CV=1.7%, Jiancheng, Nanjing, China). O2·- was

40 produced by the reaction of xanthine and xanthine oxidase. The solution was colored

41 after adding the electron acceptor and Griess reagent.

42 Antioxidant enzyme activity, lipid peroxidation and lactate dehydrogenase (LDH)

43 determination

44 Catalase (CAT) was determined based on its ability to scavenge H2O2 according to

45 the method of the catalase assay kit (A007-1-1, CV=1.7%, Jiancheng, Nanjing, China).

46 CAT could catalyze H2O2, and the reaction could be ended by adding ammonium

47 molybdate into the solution. The surplus H2O2 interacted with ammonium molybdate

48 to produce a peroxomolybdic acid complex. The absorbance was determined at 405 nm

49 by a microplate reader (Thermo, USA). One unit of CAT activity was defied as the

50 amount of enzyme to 1 μmol H2O2 per second. SOD activity was examined by a

51 superoxide dismutase assay kit (A001-3, CV=1.2%, Jiancheng, Nanjing, China), and

52 expressed as U/mg protein.

53 Malondialdehyde (MDA) can react with thiobarbituric acid (TBA) to form TBA-

54 reactive products. Lipid peroxidation in plant cells was assayed as the concentration of

55 TBA-reactive products at 535 nm, equated with MDA by the level of malondialdehyde

S3

56 (MDA) which was measured by using the lipid peroxidation MDA assay kit (S0131,

57 Beyotime Biotechnology Institute, Jiangsu, China) following the manufacturer’s

58 protocol.

59 Lactate dehydrogenase (LDH) was performed by using LDH Cytotoxicity Assay Kit

60 (C0016, Beyotime Institute of Biotechnology, Jiangsu, China). Cells were centrifuged

61 (123 ×g, 10 min) after incubation with CuO NPs (12 mg/L), CuO BPs (12 mg/L) or

62 Cu2+ (0.8 mg/L) for 4 h. The release of LDH in the supernatants was determined by

63 detecting the absorbance at 490 nm using a microplate reader (Thermo, USA).64

S4

65

66

67

68 Figure S1. TEM and SEM images of CuO NPs and BPs. (A) and (B) were the TEM images of CuO 69 NPs and BPs, respectively. (C) and (D) were the SEM images of CuO NPs and BPs, respectively.

70

710 5 10 15 20 25 30 35 40

0

0.2

0.4

0.6

0.8

1

CuO NPs

CuO BPs

Time (h)

Rel

ease

d C

u2+

(mg/

L)

72 Figure S2. The dissolution of CuO NPs and CuO BPs as a function of incubation times (0-36 h) in

73 the modified MS medium (n=3). The concentrations of CuO NPs and BPs were both 12 mg/L.

74

C D

100 nm

BA

1 μm

S5

75

0 2 4 8 16 240_

20_

40_

60_

80_

100_

120_

Time (h)

Cel

l Via

bilit

y (%

)

76 Figure S3. Cell viability after exposure to Cu2+ ions (9.6 mg/L, equal Cu content of CuO NPs at 12

77 mg/L) as a function of exposure time (0-24 h) (n=3).

78

79

80 Figure S4. High resolution TEM (HRTEM) image of intracellular CuO NPs after exposure to CuO

81 NPs (12 mg/L) for 12 h. This HRTEM image was the enlarged view of the region as marked with

82 blue box in Figure 2B.

83

5 nm

0.254 nm

CuO 002

S6

84

85

86

87 Figure S5. TEM images of BY-2 cells after exposure to CuO BPs (12 mg/L) (A) and Cu2+ (0.8

88 mg/L) (B) for 12 h. In the two panels, Cu contents marked with blue boxes were analyzed with EDS,

89 and the weight percentages of Cu content were shown along with the blue boxes. cw: cell wall; va:

90 vacuole; mit: mitochondria.

9192

A

mit

cw

B

va

mit

va

Cu, 1.43%

cw1 μm

Cu, 1.11%

200 nm

S7

C

930

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Non-inhibitor

Intr

acel

lula

r C

u (m

g C

u/g

cells

dry

wei

ght)

*

*

Control 12 mg/L2 mg/L

A B

949596979899

100101102103104105106

107 Figure S6. Internalization of CuO NPs by BY-2 cells. (A): Intracellular Cu content with/without the

108 pre-treatment of an endocytosis inhibitor (wortmannin 33 µM). After pre-treatment for 30 min, the

109 cells were exposed to CuO NPs (2, 12 mg/L) for 12 h before Cu determination. (B): TEM image of

110 plant cell after exposure to CuO NPs (12 mg/L, 12 h). (C): The elemental composition of vesicle in

111 panel B (as marked with red dashed box) was analyzed with EDS. The insert in panel (C) listed the

112 weight percentage and atom percentage of the detected elements. In panel (A), “*” indicates

113 significant differences between the “Non-inhibitor” and “Inhibitor” treatments (p < 0.05, LSD, n=3).

114

Element Weight

percentage

Atom percentage

C 75.74 90.10

O 5.80 5.18

Cl 2.54 1.02

Ni 6.08 1.48

Cu 9.85 2.21

Cu, 9.85%

S8

115

116complex III

NADH

FMN→[Fe-S]

complex I

Succinate

FAD→[Fe-S]

complex II

cyt c cyt a3

complex IV

Rotenone

TTFA

Antimycin A NaCNUbiquinone

uinone

Ubisemiquinone

Ubiquinol

Ubiquinone

117 Figure S7. Schematic diagram of mitochondrial electron transport chain and the target sites of four

118 inhibitors (rotenone, TTFA, antimycin A and NaCN) in mitochondria. Rotenone could block the

119 transfer of electrons from NADH to ubiquinone. Antimycin A can block electron transfer from

120 ubisemiquinone to ubiquinol. TTFA inhibits electron transfer from succinate to ubiquinone. NaCN

121 inhibits the oxidation of cyt a3 by O2, which could decrease oxygen production. As indicated in the

122 figure, CuO NPs blocked the electron transport from NADH to ubiquinone, and ubisemiquinone to

123 ubiquinol. Cyt: cytochrome; TTFA: thenoyltrifluoroacetone; NaCN: sodium cyanide.

124

S9

125

126

-1-2

012

CK (0 mg/L) NPs (12 mg/L)

A

6420-2-4-6log2fold change

150

100

50

0

-

log10

P-

valu

e

Filtered genesSignificant up regulated genesSignificant down regulated genesInsignificant genes

B

127 Figure S8. Hierarchical cluster (A) and volcano plot (B) for differential expression analysis of BY-2

128 cells after CuO NPs (0, 12 mg/L) exposure for 4 h. In panels A and B, red and green represent

129 significantly up-, and down-regulated genes, respectively (P-value < 0.05, fold change > 2). In panel

130 B, gray represents filtered genes, blue represents insignificantly expressed genes.

131

132

0

0.5

1

1.5

2

2.5

Expr

essi

on L

evel

*

*ControlCuO NPs

XM_016650487.1 XM_016633593.1

*

XM_016653991.1

133 Figure S9. Expression level of the three selected genes as analyzed using qRT-PCR. The plant cells

134 were exposed with CuO NPs (12 mg/L) for 12h. “*” represents significant difference at p < 0.05.

135

136

S10

137

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

ko00100: Steroid biosynthesis

ko00196: Photosynthesis − antenna proteins

ko00360: Phenylalanine metabolism

ko00362: Benzoate degradation

ko00480: Glutathione metabolism

ko00563: Glycosylphosphatidylinositol(GPI)−anchor biosynthesis

ko00591: Linoleic acid metabolism

ko00750: Vitamin B6 metabolism

ko00920: Sulfur metabolism

ko00940: Phenylpropanoid biosynthesis

ko00942: Anthocyanin biosynthesis

ko00945: Stilbenoid, diarylheptanoid and gingerol biosynthesis

ko00950: Isoquinoline alkaloid biosynthesis

ko02010: ABC transporters

ko03040: Spliceosome

ko03450: Non−homologous end−joining

ko04010: MAPK signaling pathway

ko04016: MAPK signaling pathway − plant

ko04115: p53 signaling pathway

ko04214: Apoptosis − fly

2 3 4 5Enrichment Score

Number

●●●30

0.02

0.04

0.06

P-value

10

20

138 Figure S10. Bubble chart of the top 20 KEGG pathways. The left Y-axis indicate the KEGG

139 pathways, and the X-axis indicate the respective enrichment scores. A high P-value is represented

140 by purple, and low P-value is represented by red.

141

S11

142 Table S1. The components of MS and modified MS medium (1/2 MS medium) for tobacco BY-2

143 cell culturing.

ComponentMS medium content (mg/L)

Modified MS medium (mg/L)

Macronutrients NH4NO3 1650 875KH2PO4 225 112.5KNO3 1900 975CaCl2·2H2O 440 220MgSO4·7H2O 370 185

Micronutrients KI 0.83 0.415H3BO3 6.2 3.1MnSO4·4H2O 22.3 11.15ZnSO4·7H2O 8.6 4.3Na2MoO4·2H2O 0.25 0.125CuSO4·5H2O 0.025 0.0125CoCl2·6H2O 0.025 0.0125

Iron Source EDTANa2·2H2O 37.3 18.65FeSO4·7H2O 27.8 13.9

Vitamins and Carbon Source

Myo-inositol 100 50

Glycine 2 1Thiamine-HCl 1 0.5Pyridoxine-HCl 0.5 0.25Nicotinic acid 0.5 0.25Sucrose 30000 15000

144

145

S12

146 Table S2. Results on RNA quality assessment of cells with and without CuO NPs (12 mg/L) 147 exposure. The exposure time was 4 h.

NumberRNA

concentration（μg/μL）

A260/280aVolume

(μL)Total yield (μg) RIN

CK1 0.7071 2.05 50 35.36 9.8

CK2 0.4972 1.99 50 24.86 10.0

CK3 0.6190 2.06 50 30.95 9.7

NP1 0.6894 2.01 50 34.47 10.0

NP2 0.5794 1.98 50 28.97 10.0

NP3 0.7641 2.03 50 38.21 10.0

148 a: A260/280 (the ratio of absorbance at 260 and 280 nm) is used to assess the purity of RNA. A ratio range at 1.8-

149 2.1 is accepted as “purity” for RNA. “RIN (RNA Integrity Number) ≥7” was used for subsequent gene analysis.

150

S13

151 Table S3. The primer sequences of the three selected genes during qRT-PCR, and the expression

152 level comparison of the three genes between qRT-PCR and RNA-Seq results.

Transcript -ID (N.

tabacum)

Forward primer Reverse primer P-value Log2fold change

(qRT-PCR)

Log2fold change

(RNA-Seq)

XM_016653991.1 CACTGCTCCACGCTAACA ATCATTAACTGCGTCAAGGC 1.98E-02 -0.78 -1.06

XM_016650487.1 TGGAGCAGACATTGCGAA GATTGTGGTACTCTCACTCCT 1.49E-02 0.26 1.00

XM_016633593.1 AACCTCATTCTTCACTCGTTC AGACCTTGCCGGACAATA 2.2E-03 1.06 2.01

153

154 Table S4. Characterization of CuO NPs and BPs

ParticlesPurity (%)

Surface area

(m2/g)

Size from manufacturer

(nm)

Size from TEM (nm)

Zeta potential

(mV)

Hydrodynamic diameter (nm)

CuO NPs

>99.9 12.01 <50 30-40-11.8a/-29.9b 238.4a/161.3b

CuO BPs

>99.9 0.51 5000 1500-2500-14.5a/-15.3b 2403a/1377b

155 a Measured in 1/2 MS medium, the concentrations of CuO NPs and BPs were 12 mg/L.156 b Measured in ultrapure water, the concentrations of CuO NPs and BPs were 12 mg/L.

157

158 Table S5. Information of RNA-Seq mapping results for the unexposed and CuO NPs-treated plant

159 cells.

Sample Software CK CK CK CuO NPs CuO NPs CuO NPs

Tophat 5.49 5.57 4.89 5.30 6.43 5.17Total reads (M)

Bowtie2 5.49 5.57 4.89 5.17 5.30 6.43

4.82 4.96 4.35 4.72 5.70 4.59Tophat

(87.9%) (88.9%) (89.0%) (88.9%) (88.6%) (88.9%)

4.21 4.31 3.83 4.07 4.15 4.95Total mapped reads (M)

Bowtie2(76.8%) (77.4%) (78.3%) (78.7%) (78.2%) (76.9%)

4.18 4.33 3.77 4.05 4.83 3.97Tophat

(76.2%) (77.8%) (77.2%) (76.3%) (75.1%) (76.9%)

3.91 4.03 3.59 3.79 3.87 4.61Reads mapped in proper pairs (M)

Bowtie2(71.2%) (72.3%) (73.4%) (73.3%) (73.0%) (71.8%)

160

S14

161 Table S6. The top 10 GO terms in the category of biological process, cellular component and

162 molecular function for the up-regulated genes in BY-2 cells after exposure to CuO NPs (12 mg/L)

163 for 4 h.

GO ID GO Term Category Enrichment

score

P-value

GO:0032957

GO:0042542

GO:0009408

GO:0009693

GO:0010311

GO:0009734

GO:0006970

GO:0010200

GO:0008152

GO:0009611

GO:0009705

GO:0019005

GO:0005634

GO:0005737

GO:0005802

GO:0005794

GO:0005768

GO:0005739

GO:0005887

GO:0048046

GO:0016758

GO:0016491

GO:0004674

GO:0043565

inositol trisphosphate metabolic process

response to hydrogen peroxide

response to heat

ethylene biosynthetic process

lateral root formation

auxin-activated signaling pathway

response to osmotic stress

response to chitin

metabolic process

response to wounding

plant-type vacuole membrane

SCF ubiquitin ligase complex

nucleus

cytoplasm

trans-Golgi network

Golgi apparatus

endosome

mitochondrion

integral component of plasma membrane

apoplast

transferase activity, transferring hexosyl groups

oxidoreductase activity

protein serine/threonine kinase activity

sequence-specific DNA binding

Biological process

Biological process

Biological process

Biological process

Biological process

Biological process

Biological process

Biological process

Biological process

Biological process

Cellular component

Cellular component

Cellular component

Cellular component

Cellular component

Cellular component

Cellular component

Cellular component

Cellular component

Cellular component

Molecular function

Molecular function

Molecular function

Molecular function

25.28

10.11

5.55

11.38

9.48

3.82

4.97

4.53

4.53

3.76

5.42

6.89

1.45

1.49

1.59

1.39

1.52

1.27

1.32

1.22

14.22

3.79

2.08

2.23

3.46E-06

4.05E-05

9.92E-05

1.19E-04

2.5E-04

4.92E-04

1.22E-03

1.86E-03

1.86E-03

2.14E-03

8.25E-04

8.8E-04

1.78E-03

0.021

0.120

0.132

0.136

0.157

0.192

0.230

4.66E-05

1.02E-03

6.66E-03

0.013

S15

GO:0005524

GO:0042802

GO:0030170

GO:0042803

GO:0044212

GO:0003700

ATP binding

identical protein binding

pyridoxal phosphate binding

protein homodimerization activity

transcription regulatory region DNA binding

transcription factor activity, sequence-specific

DNA binding

Molecular function

Molecular function

Molecular function

Molecular function

Molecular function

Molecular function

1.41

2.77

2.71

2.42

2.32

1.44

0.021

0.023

0.025

0.035

0.040

0.057

164

165

S16

166 Table S7. The top 10 GO terms in the category of biological process, cellular component and

167 molecular function for the down-regulated genes in BY-2 cells after exposure to CuO NPs (12 mg/L)

168 for 4 h.

GO ID GO Term Category Enrichment score P-value

GO:0009624

GO:0009873

GO:0009809

GO:0006952

GO:0042744

GO:0007165

GO:0006979

GO:0048364

GO:0006970

GO:0009611

GO:0031966

GO:0005576

GO:0005856

GO:0009579

GO:0005743

GO:0005739

GO:0031969

GO:0016021

GO:0005886

GO:0009536

GO:0042409

GO:0052689

GO:0004674

GO:0003700

GO:0043565

response to nematode

ethylene-activated signaling pathway

lignin biosynthetic process

defense response

hydrogen peroxide catabolic process

signal transduction

response to oxidative stress

root development

response to osmotic stress

response to wounding

mitochondrial membrane

extracellular region

cytoskeleton

thylakoid

mitochondrial inner membrane

mitochondrion

chloroplast membrane

integral component of membrane

plasma membrane

plastid

caffeoyl-CoA O-methyltransferase activity

carboxylic ester hydrolase activity

protein serine/threonine kinase activity

transcription factor activity

sequence-specific DNA binding

Biological process

Biological process

Biological process

Biological process

Biological process

Biological process

Biological process

Biological process

Biological process

Biological process

Cellular component

Cellular component

Cellular component

Cellular component

Cellular component

Cellular component

Cellular component

Cellular component

Cellular component

Cellular component

Molecular function

Molecular function

Molecular function

Molecular function

Molecular function

6.61

4.06

6.32

2.31

3.45

3.48

2.44

2.71

2.80

2.25

5.17

1.73

2.37

1.90

1.71

1.30

1.55

1.13

1.12

1.13

24.38

7.42

1.84

1.58

1.91

9.66E-05

1.53E-04

3.85E-04

2.17E-03

5.98E-03

0.010

0.011

0.016

0.022

0.032

2.54E-03

0.019

0.038

0.074

0.099

0.116

0.128

0.155

0.231

0.275

3.1E-06

6.3E-04

0.011

0.012

0.025

S17

GO:0016887

GO:0004601

GO:0004842

GO:0005215

GO:0044212

ATPase activity

peroxidase activity

ubiquitin-protein transferase activity

transporter activity

transcription regulatory region DNA

binding

Molecular function

Molecular function

Molecular function

Molecular function

Molecular function

2.34

2.13

1.83

2.06

1.74

0.039

0.052

0.056

0.058

0.094

169

170

S18

171 Table S8. The genes in the GO terms associated with mitochondria.

GO ID GO Term P-value Transcript ID

(N.tabacum)

Gene ID

(N. tabacum)

Log2fold

changes

Sequence

matching rate

BLAST e value Transcript ID

(Arabidopsis)

Gene Name

(Arabidopsis)

GO:

0031966

mitochondrial membrane 0.018 XM_016655108.1

XM_016658402.1

XM_016660535.1

LOC107827888

LOC107830762

LOC107832675

-3.85

-3.98

-1.10

76.54

69.2

70.81

1E-153

6E-57

2E-77

AT1G34065.1

AT3G17611.2

AT3G28580.1

SAMC2

ATRBL14

NN

GO:

0005743

mitochondrial inner

membrane

0.088 XM_016634074.1

XM_016645539.1

XM_016659246.1

LOC107809440

LOC107819429

LOC107831479

-1.48

-1.42

+/0

69.97

76.49

―

3E-66

2E-72

―

AT5G19630.1

AT1G02410.1

―

NN

COX11

NN

GO:

0005739

mitochondrion 0.075 XM_016580765.1

XM_016625037.1

XM_016637446.1

XM_016641052.1

XM_016641384.1

XM_016645539.1

XM_016653798.1

XM_016653991.1

XM_016655108.1

XM_016655916.1

XM_016656504.1

XM_016658711.1

XM_016659246.1

XM_016659608.1

XM_016660535.1

LOC107762408

LOC107801659

LOC107812378

LOC107815466

LOC107815753

LOC107819429

LOC107826771

LOC107826946

LOC107827888

LOC107828574

LOC107829073

LOC107830996

LOC107831479

LOC107831816

LOC107832675

-1.13

-2.72

-1.43

-1.58

-4.62

-1.42

-1.83

-1.06

-3.85

-2.45

-1.80

-1.30

+/0

-1.11

-1.10

70.12

72.52

64.95

74.85

70.73

76.49

70.11

79.56

76.54

67.42

―

80

―

72.38

70.81

3E-125

0

5E-29

0

0

2E-72

4E-136

1E-171

1E-153

6E-167

―

5E-09

―

0

2E-77

AT3G25210.1

AT5G22800.1

AT1G43980.1

AT4G17740.2

AT5G14220.1

AT1G02410.1

AT1G71060.1

AT3G27380.2

AT1G34065.1

AT1G53600.1

―

AT1G50270.1

―

AT5G57480.1

AT3G28580.1

NN

EMB86

PCMP-E58

CTPA2

PPOX2

COX11

NN

SDH2-1

SAMC2

PCMP-E63

NN

PCMP-E42

NN

NN

NN

GO:

0070469

respiratory chain 0.1026 XM_016634074.1 LOC107809440 -1.48 69.97 3E-66 AT5G19630.1 NN

GO:

0005749

mitochondrial respiratory

chain complex II, succinate

dehydrogenase complex

0.003 XM_016653991.1 LOC107826946 -1.06 79.56 1E-171 AT3G27380.2 SDH2-1

S19

(ubiquinone)

GO:

0008137

NADH dehydrogenase

(ubiquinone) activity

0.043 XM_016634074.1 LOC107809440 -1.48 69.97 3E-66 AT5G19630.1 NN

GO:

0010275

NAD(P)H dehydrogenase

complex assembly

0.0009 XM_016637993.1 LOC107812824 -2.88 72.92 4E-24 AT5G39210.1 CRR7

GO:

0034551

mitochondrial respiratory

chain complex III assembly

0.0003 XM_016659608.1 LOC107831816 -1.11 72.38 0 AT5G57480.1 NN

GO:

0051287

NAD binding 0.0003 XM_016660215.1 LOC107832374 -1.06 75.36 0 AT1G79750.1 NADP-ME4

GO:

0070469

respiratory chain 0.1026 XM_016634074.1 LOC107809440 -1.48 69.97 3E-66 AT5G19630.1 NN

GO:

0005739

mitochondrion 0.157 XM_016580880.1

XM_016596624.1

XM_016612125.1

XM_016628836.1

XM_016633593.1

XM_016634294.1

XM_016640170.1

XM_016641568.1

XM_016653973.1

XM_016655266.1

XM_016660604.1

LOC107762521

LOC107776706

LOC107790219

LOC107804891

LOC107809014

LOC107809633

LOC107814719

LOC107815875

LOC107826927

LOC107828015

LOC107832733

1.06

0/+

1.79

1.46

2.01

2.63

2.02

2.21

2.21

1.18

5.59

79.53

71.25

66.77

73.42

80.03

―

―

67.95

68.61

72.96

65.2

0

9E-113

2E-64

0

0

―

―

2E-118

1E-78

0

4E-58

AT3G12580.1

AT4G25290.1

AT5G38710.1

AT3G46100.1

AT1G15690.1

―

―

AT1G07590.1

AT4G27940.1

AT2G38400.1

AT5G57250.1

MED37C

NN

POX2

ATHRS1

AVP1

NN

NN

NN

MTM1

AGT3

NN

GO:

0045041

protein import into

mitochondrial

intermembrane space

0.0001 XM_016650487.1 LOC107823778 1.00 71.38 0 AT2G29080.1 FTSH3

172 “―” indicated that this gene cannot match to that of Arabidopsis. Sequence matching rate of BY-2 cells in this work was matched

173 with that of Arabidopsis.

174 “NN” in the last column indicates “No Name” of this gene was found from the genome of Arabidopsis

175 (http://www.arabidopsis.org).

S20

176 “+/0” means the gene only expressed in the unexposed cells, and this gene in the CuO NPs-treated cells was down-regulated.

177 “0/+” means the gene only expressed in the CuO NPs-treated cells, and this gene was up-regulated because it was not expressed in

178 the unexposed cells.

179

180

S21

181 Table S9. The genes that belong to glutathione metabolism in KEGG pathway analysis.

Transcript ID (N.

tabacum)

Gene ID

(N. tabacum)

Log2fold

change

Sequence

matching rate

BLAST e

value

Transcript ID

(Arabidopsis)

Gene Name

(Arabidopsis)

XM_016578004.1 LOC107759987 1.31 70.77 3.00E-77 AT5G02780.1 GSTL1

XM_016578005.1 LOC107759987 1.12 70.77 3.00E-77 AT5G02780.1 GSTL1

XM_016589828.1 LOC107770509 1.77 71.56 1.00E-83 AT5G02790.1 GSTL3

XM_016599675.1

XM_016600186.1

LOC107779283

LOC107779712

1.12

-3.70

67.77

69.9

9.00E-39

3.00E-26

AT1G02920.1

AT2G29450.1

GSTF7

GSTU5

XM_016600476.1 LOC107779974 1.42 70.39 2.00E-82 AT4G32320.1 APX6

XM_016603912.1 LOC107782951 1.66 73.42 3.00E-108 AT1G78380.1 GSTU19

XM_016603942.1

XM_016605205.1

LOC107782987

LOC107784134

1.48

-1.09

72.98

66.14

9.00E-39

1.00E-44

AT2G29420.1

AT4G39640.2

GSTU7

GGT1

XM_016611777.1

XM_016617453.1

LOC107789895

LOC107794892

1.74

-1.57

66.67

77.55

1.00E-24

0

AT3G09270.1

AT5G35790.1

GSTU8

APG1

XM_016617761.1 LOC107795177 1.20 81.25 1.00E-31 AT1G02920.1 GSTF7

XM_016621022.1

XM_016624113.1

LOC107798067

LOC107800866

1.53

1.60

68.9

69.9

3.00E-26

3.00E-26

AT2G29420.1

AT2G29450.1

GSTU7

GSTU5

XM_016641574.1 LOC107815922 -1.39 ― ― ― NN

XM_016650661.1 LOC107823949 1.37 72.86 3.00E-102 AT1G78380.1 GSTU19

XM_016659023.1 LOC107831268 1.16 67 1.00E-24 AT3G09270.1 GSTU8

XM_016660621.1 LOC107832748 1.01 71.22 0 AT5G27380.1 GSH2

XM_016640514.1 LOC107815009 -1.63 69.53 1.00E-16 AT2G29420.1 GSTU7

182 “―” indicated that this gene cannot match to that of Arabidopsis. Sequence matching rate of BY-2 cells in this work was matched

183 with that of Arabidopsis.

184 “NN” in the last column indicates “No Name” of this gene was found from the genome of Arabidopsis

185 (http://www.arabidopsis.org).